In this application, we show that the combination of the mTOR inhibitor rapamycin and the anti-androgen bicalutamide caused long-term growth inhibition and apoptosis in androgen independent prostate cancer (AIPC) cells despite neither drug having the same effect as single agents. The overall objective of the present project is to elucidate the signal transduction pathways affected by this treatment, which lead to cell cycle arrest and apoptosis. In preliminary studies, we provide novel evidence that bicalutamide alone was unable to inhibit cell growth and induce apoptosis in AIPC cells, due to the presence of a strong androgen-independent, but rapamycin-sensitive, survival pathway downstream of mTOR. On the other hand, rapamycin as monotherapy was unable to induce long-term growth inhibition due to the stimulation of an AR-dependent cell survival pathway by rapamycin treatment. Thus we hypothesize that the mTOR and AR pathways regulate survival in prostate cancer cells in parallel, and apoptosis was induced only when both pathways were simultaneously inhibited. The following specific aims have been designed to test this hypothesis: 1. To test the hypothesis that the anti-androgen bicalutamide is unable to induce apoptosis in androgen-independent prostate cancer cells due to the presence of an androgen-independent, but rapamycin-sensitive, cell survival pathway downstream of mTORC1, a complex of mTOR. mTORC1 phosphorylates downstream targets p70S6 kinase and 4E-BP1, an eIF4E binding protein. We will determine whether increased activation of the mTORC1/4E-BP1/eIF4E pathway promotes androgen-independent cell proliferation and survival in androgen-dependent prostate cancer cells. In addition, we will examine whether the mTOR pathway is activated in androgen-independent human prostate cancer tissues. 2: To test the hypothesis that long-term treatment of prostate cancer cells with the mTOR inhibitor rapamycin stimulates AR transcriptional activity resulting in increased survival and resistance to growth inhibition by rapamycin. (i) We will test the hypothesis that long-term rapamycin treatment stimulates AR transcriptional activity in prostate cancer cells. (ii) We will test the hypothesis that rapamycin-stimulated AR transcriptional activity results in increased cell survival which causes resistance to rapamycin treatment. 3. To test the hypothesis that combination treatment with rapamycin and bicalutamide induce apoptosis in prostate cancer cells and prevent AIPC. We will determine whether rapamycin and bicalutamide in combination prevent the growth of androgen-independent prostate tumors in vivo. Further, we will investigate whether combination treatment with rapamycin and bicalutamide in androgen-dependent tumors prevent the recurrence of prostate tumors in a model of prostate cancer progression. PUBLIC HEALTH RELEVANCE: This is the first time that the combination of rapamycin and bicalutamide will be used in preclinical studies for the treatment of androgen-independent prostate cancer. The combination of these drugs has not been shown before to sensitize hormone-refractory cells to hormone therapy (androgen deprivation or anti-androgens). The studies proposed here would justify a Phase II/Phase III clinical trial utilizing the combination of bicalutamide and rapamycin for the treatment of patients who experience recurrent prostate cancer. Currently, the options for patients with androgen-independent prostate cancer are limited;hence such a study would open the doors for a new treatment option.